Granted Patent
B2
US 12,496,360 · App. 18/319,042 · Granted Dec 16, 2025
Gene editing-based method of attenuating the beta-amyloid pathway
Assignee:
Wisconsin Alumni Research Foundation
A61K48/0066A61K9/0019A61K48/0008A61K48/0058A61K48/0075A61P25/28C07K14/4711C12N9/22C12N15/102C12N15/907C12N2310/20C12N2740/16043C12N2750/14143
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Abstract
Described herein are CRISPR/Cas9 constructs designed for the C-terminal truncation of human amyloid precursor protein (APP) as well as methods of making and using such a construct.
Claims (8)
1 . A method of pre-treating a patient at risk of having or acquiring Alzheimer's disease (AD) caused by formation of amyloid plaques composed of amyloid beta (AB) peptides, wherein the method comprises the steps of
(a) obtaining a gene-editing construct specific for the amyloid precursor protein (APP), wherein the gene-editing construct facilitates truncation of the APP C-terminus when combined with a Cas9 nuclease, and
(b) delivering the gene-editing construct and a construct encoding the Cas9 nuclease to a patient in need of AD therapy, wherein the APP molecule is truncated and production of Aβ peptides is decreased in the patient's brain, wherein the truncation of the APP C-terminus occurs at an APP residue selected from the group consisting of 659, 670, 676, and 686 relative to SEQ ID NO: 12 (human) or SEO ID NO: 14 (mouse).
2 . The method of claim 1 , wherein the gene-editing construct comprises a gRNA sequence selected from the group consisting of SEQ ID NOs: 1-10.
3 . The method of claim 1 , wherein the gene-editing construct and the construct encoding the Cas9 nuclease are delivered in a composition comprising an adeno-associated viral vector and a nanocarrier delivery vehicle.
4 . The method of claim 3 , wherein the composition is delivered intravenously or intrathecally.
5 . The method of claim 1 , wherein the patient has a genetic risk factor for AD or a family history of AD.
6 . The method of claim 5 , wherein the patient has an APPV717I mutation.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded May 25, 2023
From: SUN, JICHAO; ROY, SUBHOJIT
To: WISCONSIN ALUMNI RESEARCH FOUNDATION
Reel/Frame 063758/0083 →
Continuity (4)
Provisional Application
62618694
· Jan 18, 2018
References Cited (85)
WO 0173002A2
· 2001
[cited by applicant]
Aime, P. et al.; Trib3 Is Elevated in Parkinson's Disease and Mediates Death in Parkinson's Disease Models: Journal of Neuroscience, vol. 35; 2015; pp. 10731-10749, doi:10.1523/JNEUROSCI.0614-15.2015.
[cited by applicant]
Banks, W.; “From blood-brain barrier to blood-brain interface: new opportunities for CNS drug delivery”; Nature Reviews, vol. 15; 2016; pp. 275-292.
[cited by applicant]
Beckett, C., et al.; Nuclear signalling by membrane protein intracellular domains: the AICD enigma; Cell Signal, vol. 24; 2012; pp. 402-409; doi:10.1016/j.cellsig.2011.10.007.
[cited by applicant]
Brinkman, E. K. et al.; “Easy quantitative assessment of genome editing by sequence trace decomposition”; Nucleic Acids Research vol. 42, e168; 2014; doi:10.1093/nar/gku936.
[cited by applicant]
Carlson-Stevermer, J. et al. “Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing”; Nat Commun, vol. 8, 1711; 2017; doi: 10.1038/s41467-017-01875-9.
[cited by applicant]
Chakrabarty, P. et al.; “Capsid serotype and timing of injection determines AAV transduction in the neonatal mice prain”; PLoS One, vol. 8, e67680; 2013; doi:10.1371/journal.pone.0067680.
[cited by applicant]
Chow, V. W., et al.; “An overview of APP processing enzymes and products” Neuromolecular Med, vol. 12; 2010; pp. 1-12; doi:10.1007/s12017-009-8104-z.
[cited by applicant]
Citron, M. et al.; “Generation of amyloid beta protein from its precursor is sequence specific”; Neuron, vol. 14; 1995; pp. 661-670; doi:0896-6273(95)90323-2.
[cited by applicant]
Das, U. et al.; “Activity-induced convergence of APP and BACE-1 in acidic microdomains via an endocytosis-dependent pathway”; Neuron, vol. 79; 2013; pp. 447-460, doi:10.1016/j.neuron.2013.05.035.
[cited by applicant]
Das, U. et al.; “Visualizing APP and BACE-1 approximation in neurons yields insight into the amyloidogenic pathway” Nat Neurosci, vol. 19; 2016; pp. 55-64; doi:10.1038/nn.4188.
[cited by applicant]
De Strooper, B. et al.; “The Cellular Phase of Alzheimer's Disease”; Cell, vol. 164; 2016; pp. 603-615.
[cited by applicant]
Deverman, B. et al.; “Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain”; Nature Biotechnology, vol. 34, Issue No. 2; 2016; pp. 204-209.
[cited by applicant]
Deyts, C. et al.; “APP Receptor? To Be or Not To Be”; Trends in Pharmacological Sciences, vol. 37; 2016; pp. 390-411, doi:10.1016/j.tips.2016.01.005.
[cited by applicant]
Doench, J. et al.; “Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9”; Nature Biotechnology, vol. 34, Issue No. 2; 2016; pp. 184-191 doi:10.1037/nbt3437.
[cited by applicant]
Fellmann, C. et al.; “Cornerstones of CRISPR-Cas in drug discovery and therapy”; Nat Rev Drug Discov, vol. 16; 2017; pp. 89-100; doi:10.1038/nrd.2016.238.
[cited by applicant]
Fol, R. et al. “Viral gene transfer of APPsalpha rescues synaptic failure in an Alzheimer's disease mouse model”; Acta Neuropathol, vol. 131; 2016; pp. 247-266; doi:10.1007/s00401-015-1498-9.
[cited by applicant]
Fu, Y. et al.; “Improving CRISPR-Cas nuclease specificity using truncated guide RNAs”; Nature Biotechnology, vol. 32, Issue No. 3; 2014; pp. 279-284.
[cited by applicant]
Guo, W. et al.; “Fragile X Proteins FMRP and FXR2P Control Synaptic GluA1 Expression and Neuronal Maturation via Distinct Mechanisms”; Cell Rep, vol. 11; 2015; pp. 1651-1666; doi:10.1016/j.celrep.2015.05.013.
[cited by applicant]
Gyorgy, B. et al.; “CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer's Disease”; Mol Ther Nucleic Acids, vol. 11; 2018; pp. 429-440, doi:10.1016/j.omtn.2018.0…
[cited by applicant]
Haass, C. et al.; “Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid β-peptide”; Molecular Cell Biology, vol. 8; 2007; pp. 101-112.
[cited by applicant]
Haass, C. et al.; “Trafficking and Proteolytic Processing of APP”; Cold Spring Harbor Perspectives in Medicine, vol. 2, Issue No. 5; 2012; pp. 1-25.
[cited by applicant]
Hampel, H. et al.; “The future of Alzheimer's disease: The next 10 years”; Progress in Neurobiology, vol. 95; 2011; pp. 718-728.
[cited by applicant]
Hardy, J. et al.; “The Amyloid Hypothesis of Alzheimer's Disease: Progress and Problems on the Road to Therapeutics”; Science, vol. 297; 2002; pp. 353-355.
[cited by applicant]
Hendriks, L. et al.; “Presenile dementia and cerebral haemorrhage linked to a mutation at codon 692 of the β-amyloid precursor protein gene”; Nature Genetics, vol. 1; 1992; pp. 218-221.
[cited by applicant]
Hocquemiller, M. et al.; “Adeno-Associated Virus-Based Gene Therapy for CNS Diseases”; Human Gene Therapy, vol. 27, Issue No. 7; 2016; pp. 478-496.
[cited by applicant]
International Search Report and Written Opinion for International Application PCT/US2019/014249; International Filing Date: Jan. 18, 2019; Date of Mailing: May 21, 2019; 16 pages.
[cited by applicant]
Joung, J. et al.; “Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening”; Nat Protoc, vol. 12; 2017; pp. 828-863; doi:10.1038/nprot.2017.016.
[cited by applicant]
Kim, J. Y.; “Widespread Neuronal Transduction of the Rodent CNS via Neonatal Viral Injection”; Methods Mol Biol, vol. 1382; 2016; pp. 239-250; doi:10.1007/978-1-4939-3271-9_17.
[cited by applicant]
Komor, A. et al.; “CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes”; Cell, vol. 168; 2017; pp. 20-36.
[cited by applicant]
Koo, E. H. et al.; “Evidence that production and release of amyloid beta-protein involves the endocytic pathway”; J Biol Chem, vol. 269; 1994; pp. 17386-17389.
[cited by applicant]
Kuscu, C. et al.; “Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease”; Nature Biotechnology, vol. 32, Issue No. 7; 2014; pp. 677-683.
[cited by applicant]
Lai, A., et al.; “Characterization of sorting signals in the beta-amyloid precursor protein cytoplasmic domain”; J Biol Chem, vol. 270; 1995; pp. 3565-3573.
[cited by applicant]
Lee, M. S. et al.; “APP processing is regulated by cytoplasmic phosphorylation”; J Cell Biol, vol. 163; 2003; pp. 83-95; doi:10.1083/jcb.200301115.
[cited by applicant]
Liu, Y. et al.; “Brain-targeting gene delivery and cellular internalization mechanisms for modified rabies virus glycoprotein RVG29 nanoparticles”; Biomaterials, vol. 30; 2009; pp. 4195-4202.
[cited by applicant]
Luo, S. et al.; “Water Soluble Poly(histamine acrylamide) with Superior Buffer Capacity Mediates Efficient and Nontoxic In Vitro Gene Transfection”; Journal of Polymer Science Part A: Polymer Chemistry, vol. 49; 2011; p…
[cited by applicant]
McMahon, M. A. et al.; “Gene therapy: Gene-editing therapy for neurological disease”; Nat Rev Neurol, vol. 13; 2017; pp. 7-9; doi:10.1038/nmneurol.2016.190.
[cited by applicant]
Mendell, J. et al.; “Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy”; The New England Journal of Medicine, vol. 377, Issue No. 18; 2017; pp. 1713-1722.
[cited by applicant]
Mockett, B. G. et al.; “Therapeutic Potential of Secreted Amyloid Precursor Protein APPsalpha”; Front Mol Neurosci, vol. 10; 2017; pp. 30; doi:10.3389/fnmol.2017.00030.
[cited by applicant]
Morel, E. et al.; “Phosphatidylinositol-3-phosphate regulates sorting and processing of amyloid precursor protein through the endosomal system”; Nat Commun, vol. 4, Issue No. 2250; 2013; doi:10.1038/ncomms3250.
[cited by applicant]
Muller, U. C. et al.; “Physiological functions of APP family proteins”; Cold Spring Harb Perspect Med, vol. 2, a006288; 2012; doi:10.1101/cshperspect.a006288.
[cited by applicant]
Muller, U. C., et al.; “Not just amyloid: physiological functions of the amyloid precursor protein family”; Nat Rev Neurosci, vol. 18; 2017; pp. 281-298, doi:10.1038/nrn.2017.29.
[cited by applicant]
Musiek, E. et al.; “Three dimensions of the amyloid hypothesis: time, space and ‘wingmen’”; Nature Neuroscience, vol. 18; 2015; pp. 800-806.
[cited by applicant]
Nitsch, et al.; “Release of Alzheimer amyloid precursor derivatives stimulated by activation of muscarinic acetylcholine receptors”; Science, vol. 258; 1992; pp. 304-307.
[cited by applicant]
O'Brien, R. J. et al.; “Amyloid precursor protein processing and Alzheimer's disease”; Annu Rev Neurosci, vol. 34; 2011; 185-204, doi:10.1146/annurev-neuro-061010-113613.
[cited by applicant]
Paquet, D. et al.; “Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9”; Nature, vol. 533, 125-129; 2016; doi:10.1038/nature17664.
[cited by applicant]
Pardossi, et al.; “The physiology of the beta-amyloid precursor protein intracellular domain AICD”; J Neurochem, vol. 120, Suppl 1; 2012; pp. 109-124; doi:10.1111/j.1471-4159.2011.07475.x.
[cited by applicant]
Park, C. Y. et al.; “Reversion of FMR1 Methylation and Silencing by Editing the Triplet Repeats in Fragile X iPSC-Derived Neurons”; Cell Rep, vol. 13; 2015; pp. 234-241; doi:10.1016/j.celrep.2015.08.084.
[cited by applicant]
Passini, M. A. et al.; “Widespread gene delivery and structure-specific patterns of expression in the brain after intraventricular injections of neonatal mice with an adeno-associated virus vector”; J Virol, vol. 75; 20…
[cited by applicant]
Perez, R. G. et al.; “Mutagenesis identifies new signals for beta-amyloid precursor protein endocytosis, turnover, and the generation of secreted fragments, including Abeta42”; J Biol Chem, vol. 274; 1999; pp. 18851-188…
[cited by applicant]
Perrin, et al.; “Multimodal techniques for diagnosis and prognosis of Alzheimer's disease”; Nature, vol. 461, Issue No. 7266; pp. 916-922, (2009).
[cited by applicant]
Putnam, D. et al.; “Polymer-based gene delivery with low cytotoxicity by a unique balance of side-chain termini”; PNAS, vol. 98, Issue No. 3; 2001; pp. 1200-1205.
[cited by applicant]
Ran, F. et al.; “Genome engineering using the CRISPR-Cas9 system”; Nature Protocols, vol. 8, Issue No. 11; 2013; pp. 2281-2308.
[cited by applicant]
Richter, M. C. et al.; “Distinct in vivo roles of secreted APP ectodomain variants APPsalpha and APPsbeta in regulation of spine density, synaptic plasticity, and cognition”; EMBO J, vol. 37; 2018; doi:10.15252/embj.201…
[cited by applicant]
Ring, S. et al.; “The secreted beta-amyloid precursor protein ectodomain APPs alpha is sufficient to rescue the anatomical, behavioral, and electrophysiological abnormalities of APP-deficient mice”; J Neurosci, vol. 27;…
[cited by applicant]
Rohn, T. et al.; “The Potential of CRISPR/Cas9 Gene Editing as a Treatment Strategy for Alzheimer's Disease”; Journal of Alzheimer's Disease and Parkinsonism, vol. 8, Issue No. 3; 2018; 12 pages; doi:10.4172/2161-0460.1…
[cited by applicant]
Sander, J. D. et al.; “CRISPR-Cas systems for editing, regulating and targeting genomes”; Nat Biotechnol, vol. 32; 2014; pp. 347-355; doi:10.1038/nbt.2842.
[cited by applicant]
Sanjana, N. E., et al.; “Improved vectors and genome-wide libraries for CRISPR screening”; Nat Methods, vol. 11; 2014; pp. 783-784; doi:10.1038/nmeth.3047.
[cited by applicant]
Schwartz, M. et al.; “Human pluripotent stem cell-derived neural constructs for predicting neural toxicity”; PNAS, vol. 112, Issue No. 40; 2015; pp. 12516-12521.
[cited by applicant]
Scott, D. et al.; “A Pathologic Cascade Leading to Synaptic Dysfunction in α-Synuclein-Induced Neurodegeneration”; The Journal of Neuroscience, vol. 30, Issue No. 24; 2010; pp. 8083-8095.
[cited by applicant]
Scott, D. et al.; “Mechanistic Logic Underlying the Axonal Transport of Cytosolic Proteins”; Neuron, vol. 70, Issue No. 3; 2011; pp. 441-454.
[cited by applicant]
Shrestha, R. et al.; “Endosomal escape and siRNA delivery with cationic shell crosslinked knedel-like nanoparticles with tunable buffering capcities”; Biomaterials, vol. 33, Issue No. 33; 2012; pp. 8557-8568.
[cited by applicant]
Sisodia, S. S.; “Beta-amyloid precursor protein cleavage by a membrane-bound protease”; Proc Natl Acad Sci U S A, vol. 89; 1992; pp. 6075-6079.
[cited by applicant]
Sun, J. et al.; “CRISPR/Cas9 editing of APP C-terminus attenuates β-cleavage and promotes α-cleavage”; Nature Communications, vol. 10, Issue No. 1; 2019; 11 pages; doi:10.1038/s41467-018-07971-8.
[cited by applicant]
Sun, J. et al.; “The physical approximation of APP and BACE-1: A key event in alzheimer's disease pathogenesis”; Dev Neurobiol, vol. 78; 2018; pp. 340-347, doi:10.1002/dneu.22556.
[cited by applicant]
Swiech, L. et al.; “In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9”; Nat Biotechnol, vol. 33; 2015; pp. 102-106; doi:10.1038/nbt.3055.
[cited by applicant]
Tang, Y. et al.; “Early and selective impairments in axonal transport kinetics of synaptic cargoes induced by soluble amyloid beta-protein oligomers”; Traffic , vol. 13; 2012; pp. 681-693; doi:10.1111/j.1600-0854.2012.0…
[cited by applicant]
Thinakaran, G. et al.; “Amyloid precursor protein trafficking, processing, and function”; J Biol Chem, vol. 283; 2008; pp. 29615-29619; doi:10.1074/jbc.R800019200.
[cited by applicant]
Topol, A., et al.; “A guide to generating and using hiPSC derived NPCs for the study of neurological diseases”; J Vis Exp, e52495; 2015; doi:10.3791/52495.
[cited by applicant]
Ubelmann, F. et al.; “Bin1 and CD2AP polarise the endocytic generation of beta-amyloid”; EMBO Rep, vol. 18; 2017; pp. 102-122, doi:10.15252/embr.201642738.
[cited by applicant]
Vassar, R. et al.; “Function, therapeutic potential and cell biology of BACE proteases: current status and future prospects”; J Neurochem, vol. 130; 2014; pp. 4-28, doi:10.1111/jnc.12715.
[cited by applicant]
Vassar, R. et al.; “The β-Secretase Enzyme BACE in Health and Alzheimer's Disease: Regulation, Cell Biology, Function, and Therapeutic Potential”; The Journal of Neuroscience, vol. 29, Issue No. 41; 2009 pp. 12787-12794.
[cited by applicant]
Veres, A. et al.; “Low Incidence of Off-Target Mutations in Individual CRISPR-Cas9 and TALEN Targeted Human Stem Cell Clones Detected by Whole-Genome Sequencing”; Cell Stem Cell, vol. 15, Issue No. 1; 2014; pp. 27-30.
[cited by applicant]
Vickers, J. et al.; “A Vaccine Against Alzheimer's Disease: Developments to Date”; Drugs and Aging, vol. 19, Issue No. 7; 2002; pp. 487-494.
[cited by applicant]
Wang, L. et al.; “α-Synuclein Multimers Clusters Synaptic Vesicles and Attenuate Recycling”; Current Biology: CB, vol. 24, Issue No. 19; 2014; pp. 2319-2326.
[cited by applicant]
Wiley, D. et al.; “Transcytosis and brain uptake of transferrin-containing nanoparticles by tuning avidity to transferrin receptor”; PNAS, vol. 110, Issue No. 21; 2013; pp. 8662-8667.
[cited by applicant]
Yang, S. et al.; “CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington's disease”; J Clin Invest, vol. 127; 2017; pp. 2719-2724; doi:10.1172/JCI92087.
[cited by applicant]
Zeitler, B. et al.; “Sustained Tau Reduction via Zinc Finger Protein Transcription Factors as a Potential Next-Generation Therapy for Alzheimer's Disease and Other Tauopathies”; available online at https://d1io3yog0oux5…
[cited by applicant]
Alzheimer's Association Report: 2014 Alzheimer's disease facts and figures, Alzheimer's & Dementia, 10 (2014) e47-e92, section 2.2.4.2.
[cited by applicant]